Colorectal cancer is the fourth most frequent cancer in the Western world, with about 160,000 new cases yearly in the US. Forty to 50% of all colorectal cancer patients will be diagnosed with early stage disease (Dukes' stage A or B). Most of these patients with early stage colorectal cancer can be cured by surgery alone. Thus, risk of recurrence is closely related to stage of disease at time of primary surgery, with about a 10% relapse rate in Dukes' stage A and 25-30% in Dukes' stage B. Patients with Dukes' stage C colorectal cancer have a five-year relapse rate of 70% following surgery and are offered adjuvant chemotherapy. Following relapse, the risk of dying of the disease is significant. Thus, one way to improve survival is to increase the number of patients being diagnosed with early stage disease. Screening for colorectal cancer has been shown to improve survival, however, current tests suffer from a lack of compliance, from low sensitivity, and from the need for strict dietary restrictions. Thus, the development of new and improved tests for the early detection of colorectal cancer is needed.
Thus, 30-40 percent of patients who have undergone a complete resection of a colorectal malignancy will experience relapse with metastatic disease, which is usually fatal. Thus, hundreds of thousands of people with resected CRC are candidates for surveillance, as it is a commonly held clinical view that early detection of metastases and metachronous disease by relevant surveillance regimens may allow for interventions with the aim of improving overall survival, disease-free survival and quality of life.
The majority of recurrences of CRC occur within 5 years, and usually within 3 years following surgery. A tremendous effort has been put into the establishment of effective tests for the detection of recurrent disease at a stage when intervention is not futile. Carcinoembryonic antigen (CEA) tests, colonoscopies, chest x-rays, liver function tests, complete blood cell counts, fecal occult blood tests, computerized tomography (CT), ultrasonography, magnetic resonance (MR) and positron emission tomography (PET) are all methods that have been extensively reported on for the postoperative surveillance of CRC. Unfortunately, the results from randomised studies of such monitoring of CRC patients have varied widely and demonstrated minimal efficacy (Schoemaker, Gastroent, 1998; Ohlsson, Dis Col Rec, 1995; Makela, Arch Surg, 1996; Kjeldsen, Int J Color Dis, 1997). As a result hereof, considerable variation in clinical follow-up practice is evident (Virgo KS, Ann Surg, 1995) and expenses for 5-year follow-up of CRC patients have differed from $561 to $16,492 per patient (Virgo, JAMA, 1995).
The American Society of Clinical Oncology (ASCO) has recommended against the use of liver function tests, fecal occult blood tests, complete blood counts, pelvic imaging, CT scanning and chest x-rays as means for regular postoperative monitoring of CRC. However, it was recommended that postoperative measurement of serum CEA is performed every 2 to 3 months for ≧2 years in patients with stage II or III disease where resection of liver metastases is clinically indicated (Benson, JCO, 2000). Of note, however, is the fact that approximately 30 percent of all CRC recurrences do not produce CEA (Safi, Cancer Detect Prev, 1993). Thus, it was stressed by the ASCO that new surveillance methods for the detection of CRC recurrences are needed (Benson, JCO, 2000). This statement has gained further weight as recent data has shown that chemotherapy of metastatic CRC can improve short-term survival and often improve the quality of life (Cunningham, Lancet, 1998) and that treatment of asymptomatic CRC yields better results than postponing treatment until the disease has become symptomatic (Nordic Gastr Tumor Adj Group, JCO, 1992).
Because metastatic disease is the main cause of cancer patient morbidity and mortality, molecules involved in the regulation of tumor invasion and metastasis are attractive as potential diagnostic/prognostic/monitoring targets. It is well established that proteolytic enzymes produced by cancer cells or by cells in the tumor stroma are involved in extracellular tissue degradation, leading to cancer cell invasion and metastasis. A number of enzymes have been associated with this process, the most thoroughly investigated being the metalloproteinases, such as the collagenases and stromelysins, and the serine proteases such as plasmin. Recently, data have been published indicating that these molecules, free or bound in complexes, are released from tumor tissue and find their way into the circulation.
Matrix metalloproteinases (MMP's) play a pivotal role in cancer growth and spread, contributing to enzymatic degradation of the extracellular matrix (Liotta et. al., 1991; Stetler-Stevenson et. al., 1993; MacDougall & Matrisian, 1995). The naturally occurring inhibitors of MMP's, tissue inhibitors of MMP's (TIMP's), form tight 1:1 stoichiometric complexes with the activated forms of the MMP's (Welgus et. al., 1985; Kleiner et. al., 1993), thereby inhibiting the catalytic activity of these enzymes (Stetler-Stevenson et. al., 1996; Goldberg et. al., 1992; Birkedal-Hansen et. al., 1993). While the balance between the matrix-degrading properties of MMP's and the inhibitory effect of TIMP's is closely regulated under normal physiological conditions (Matrisian, 1992; Thorgeirsson et. al., 1993; Birkedal-Hansen et. al., 1993), this balance might be disrupted in malignant tissue.
A number of enzyme-linked immunoassays for the detection of TIMP-1 (Kodama et. al., 1989; Cooksley et. al., 1990; Clark et. al., 1991) and TIMP-2 (Fujimoto et. al., 1993) have been described. These assays have been applied to body fluids, e.g. serum, plasma, amniotic fluid, cerebrospinal fluid, urine, but the number of samples tested has not been sufficient to establish normal ranges for TIMP levels in healthy individuals (Kodama et. al., 1989; Clark et. al., 1991). Furthermore, none of these assays have been sufficiently validated for technical performance or for clinical use. We have recently described and validated an ELISA for the quantitation of total TIMP-1 in plasma samples, and using this assay we could demonstrate that healthy blood donors have a very narrow range of total plasma TIMP-1 (Holten-Andersen et. al., Br. J. Cancer 1999). We have also recently developed and validated an ELISA for the quantitation of uncomplexed TIMP-1 in plasma (Holten-Andersen et. al., manuscript submitted)
In a study by Mimori et. al. (Mimori et. al., 1997) in which tumor tissue levels of TIMP-1 mRNA were studied in patients with gastric carcinoma, high tumor/normal tissue ratios of TIMP-1 mRNA were found to be associated with increased invasion and poor prognosis and Guillem et. al., (Proc Annu Meet Am Assoc Cancer Res; 34:A466, 1993) suggest a possible correlation between TIMP-1 RNA levels and poorly differentiated, invasive colorectal cancers. However, TIMP-1 protein levels in sera from prostate cancer patients and healthy donors (Baker et. al., 1994) showed a high degree of overlap. Similarly, a separate study of plasma from prostate cancer patients and healthy donors showed no difference in TIMP-1 levels between the two groups (Jung et al., 1997).
We have recently published that preoperatively measured plasma levels of total TIMP-1 show highly significant association with colorectal cancer patient survival, i.e. patients with high TIMP-1 levels had a significantly shorter survival than those patients with low TIMP-1 levels (Holten-Andersen et. al., Clin. Cancer Res, 2000).
Furthermore, Nauro et. al., (J. Urol, vol. 1, no 3, September 1994, pp 228-231) found that serum TIMP-1 levels are significantly higher in metastatic bladder cancer patients compared to a healthy control group, whereas no results were presented about the TIMP-1 levels of pre-metastatic bladder cancers. TIMP-1 measurements in urine have also been described as being useful in the diagnosis of other cancers such as bladder cancer, liver cancer and renal cancer (Japanese patent application no. 8-136548). In none of these studies a correlation between the TIMP-1 level of patients with early stage bladder cancers and the TIMP-1 level of patients with cystitis have been included.
Measurements of TIMP-1 levels have also been shown to be useful in diagnosing diseases of the nervous system as described in U.S. Pat. No. 5,324,634.
Studies of TIMP-1 complexed with MMP-9 in plasma of patients with advanced gastrointestinal and gynaecological cancer (Zucker et al., 1995) demonstrated significantly higher levels in blood samples from cancer patients with metastatic disease compared to healthy control individuals, and that patients with high levels of TIMP-1:MMP-9 complex had a shorter survival (Zucker et. al., 1995 and U.S. Pat. No. 5,324,634). However, this study did not include measurements of total or free TIMP-1, only the complex between TIMP-1 and one of the up to now approximately 24 identified MMP's. Furthermore, in this study, no differences in complex levels were found between patients with breast cancer and healthy donors. Also, this study did not include patients with early stage cancer.